A new experimental method for mapping secondary-structure along the primary sequence of any RNA molecule, that employs enzymes as structure probes, is used to determine the exact location of helical double-stranded regions in rabbit and human alpha and beta globin messenger RNAs. Physico-chemical parameters, e.g. temperature, pH, ionic strength, are varied to determine their effect on mRNA structure using this method. Tertiary structure information is also obtained. The method, developed and being refined in this laboratory using transfer RNA and 5s ribosomal RNA from several species as model systems, combines the following procedures: a) the use of tobacco acid pyrophosphatase to remove, specifically, the 5'-terminal 7meG "cap" nucleotide from mRNA; b) specifically 5'-end labeling "de-capped" molecules with (32P)using T4 polynucleotide kinase; c) partial hydrolysis of 5'-end labeled mRNA with a variety of ribonucleases, with OH and with several single-strand specific nucleases, such as S1, P1 and N. crassa enzymes; and d) analysis of digestion products by high-resolution polyacrylamide-urea gel electrophoresis to yield the exact location, in primary sequence, of those nucleotides accessible to the single-strand specific enzymes. A computer program capable of predicting secondary structure from nucleotide sequence is in use. This program, based on thermodynamic principles, has predicted that similar structures exist for a large number of eukaryotic and viral mRNAs in their 5'-regions. This suggests that structure is conserved in the 5'-region and may play an important role in mRNA function. The structure mapping method will be used to test these and other computer predictions, initially for the globin mRNAs. Such information will be fundamental to future considerations of the relationship of the secondary structure of eukaryotic messenger RNA to its biological function and stability.